Compositions and methods for modifying a plant characteristic without modifying the plant genome

Abstract

The invention relates to methods and compositions for modifying a characteristic of a plant without modifying the plant's genome using one or more cells comprising one or more phytohormone genes and at least one polynucleotide of interest, which one or more phytohormone genes and the at least one polynucleotide of interest are expressed in the one or more cells.

That which is claimed is: 
     
         1 . A symbiont forming inoculum comprising a polynucleotide encoding a phytohormone biosynthetic enzyme and a polynucleotide of interest, wherein the phytohormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and/or an auxin biosynthetic enzyme. 
     
     
         2 . The symbiont forming inoculum of  claim 1 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest are comprised in a cell, optionally wherein the cell is a plant cell or a bacterial cell. 
     
     
         3 . The symbiont forming inoculum of  claim 1  or  claim 2 , wherein the phytohormone biosynthetic enzyme is from a bacterial species and/or a plant species. 
     
     
         4 . The symbiont forming inoculum of any one of  claims 1 - 3 , wherein the phytohormone biosynthetic enzyme is an indole-3-acetamide hydrolase (iaaH) (E.C. Number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), a tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), an indole-3-lactate synthase (EC 1.1.1.110), a L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), a tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), a tryptophan decarboxylase 1/tryptophan decarboxylase 2 (EC4.1.1.105), an isopentenyl transferase (Ipt) and/or a Tzs (EC 2.5.1.27). 
     
     
         5 . The symbiont forming inoculum of any one of  claims 1 - 4 , wherein the phytohormone biosynthetic enzyme is an indole-3-acetamide hydrolase (iaaH), a tryptophan 2-monooxygenase (IaaM), and/or an isopentenyl transferase (Ipt). 
     
     
         6 . The symbiont forming inoculum of any one of  claims 1 - 5 , wherein the phytohormone biosynthetic enzyme is indole-3-lactate synthase. 
     
     
         7 . The symbiont forming inoculum of any one of  claims 1 - 6 , further comprising a polynucleotide encoding a plast polypeptide (e.g., plasticity polypeptide), optionally wherein the plast polypeptide is 6b, rolB, rolC, and/or orf13. 
     
     
         8 . The symbiont forming inoculum of any one of  claims 1 - 7 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest are comprised in a single nucleic acid construct or in two or more nucleic acid constructs (e.g., one or more expression cassettes). 
     
     
         9 . The symbiont forming inoculum of  claim 7  or  claim 8 , wherein the polynucleotide encoding a plast polypeptide is comprised in a nucleic acid construct, optionally wherein the polynucleotide encoding a plast polypeptide is in the same or a separate nucleic acid construct (e.g., expression cassette) as/from the polynucleotide encoding a phytohormone biosynthetic enzyme and/or the polynucleotide of interest. 
     
     
         10 . The symbiont forming inoculum of any one of  claim 8  or  claim 9 , wherein the one or more nucleic acid constructs are comprised in one or more vectors. 
     
     
         11 . The symbiont forming inoculum of  claim 10 , wherein the one or more vectors are a plasmid, a T-DNA, a bacterial artificial chromosome, viral vector, or a binary-bacterial artificial chromosome. 
     
     
         12 . The symbiont forming inoculum of any one of  claims 1 - 11 , wherein the polynucleotide of interest encodes a biomolecule, a bioactive molecule and/or a polypeptide involved in the biosynthesis of a bioactive molecule. 
     
     
         13 . The symbiont forming inoculum of  claim 12 , wherein expression of the polynucleotide of interest confers increased abiotic stress resistance (e.g., high salt tolerance, high heat tolerance, heavy metals tolerance, cold tolerance, drought tolerance, excessive water tolerance, tolerance to UV radiation), increased resistance or tolerance to a pathogen (e.g., viral, fungal, bacterial) or pest (e.g., insect, nematode), or increased tolerance to an herbicide, 
     
     
         14 . The symbiont forming inoculum of  claim 12  or  claim 13 , wherein the bioactive molecule is a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein/peptide (e.g., a bioinsecticide; e.g., jaburetox (peptide JBTX; a bioinsecticide derived from Jack beans ( Canavalia ensiformis  seeds)); insecticidal peptides from spider venom (e.g., from  Hadronyche versuta ); trypsin modulating oostatic factor (TMOF);  Bacillus thuringiensis  toxins (δ endotoxins, e.g., Cry toxin, Cyt toxin); a vegetative insecticidal protein (Vip)), a nutrient (e.g., nitrogen, e.g., a leghemoglobin, a nitrogenase), a plant growth regulator (auxin, cytokinin, gibberellin, ethylene; growth inhibitor/retardant), a plant lipid, a plant fatty acid, a plant oil, an RNA (e.g., siRNA, dsRNA, miRNA, shRNA), a plantibody, a stylet sheath inhibitory protein (e.g., ficin, bromelain), a ribozyme, a bacteriocin, an antimicrobial peptide (e.g., oncocin), an aptamer, a nuclease, a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN) and/or an engineered meganuclease. 
     
     
         14 . The symbiont forming inoculum of any one of  claims 1 - 13 , wherein the polynucleotide of interest encodes a polypeptide operably linked to a targeting sequence, optionally wherein the targeting sequence locates the protein to a membrane, a subcellular location or an extracellular location. 
     
     
         15 . The symbiont forming inoculum of  claim 14 , wherein the targeting sequence is a membrane targeting sequence, an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence, nuclear targeting sequence, vacuolar targeting sequence, peroxisomal targeting sequence, lysosomal targeting sequence, or a plant virus movement protein. 
     
     
         16 . The symbiont forming inoculum of any one of  claims 1 - 15 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme (e.g., the polynucleotide encoding iaaH, the polynucleotide encoding IaaM, and/or the polynucleotide encoding isopentenyl transferase (Ipt), and/or the polynucleotide encoding indole-3-lactate synthase) and/or the polynucleotide encoding a plast polypeptide is/are operably linked to a nuclear targeting (nuclear localization) sequence. 
     
     
         17 . The symbiont forming inoculum of any one of  claims 1 - 16 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest are each operably linked to a single promoter or to at least two separate promoters, in any combination. 
     
     
         18 . The symbiont forming inoculum of any one of  claims 1 - 17 , wherein when the polynucleotide encoding a phytohormone biosynthetic enzyme encodes iaaH, IaaM and Ipt, the polynucleotide(s) encoding iaaH, IaaM and Ipt is operably linked to a single promoter and the polynucleotide of interest is operably linked to the single promoter or to a separate promoter. 
     
     
         19 . The symbiont forming inoculum of any one of  claims 7 - 18 , wherein the polynucleotide encoding a plast polypeptide is operably linked to a promoter, optionally the polynucleotide encoding a plast polypeptide is operably linked to the same promoter or a separate promoter as that which is operably linked to the polynucleotide encoding a phytohormone biosynthetic enzyme and/or the polynucleotide of interest. 
     
     
         20 . The symbiont forming inoculum of any one of  claims 17 - 19 , wherein the single promoter, the separate promoter, and/or the two or more separate promoters are each a constitutive promoter or an inducible promoter in any combination. 
     
     
         21 . The symbiont forming inoculum of any one of  claims 1 - 20 , wherein the polynucleotide of interest is expressed in the symbiont forming inoculum. 
     
     
         22 . The symbiont forming inoculum of any one of  claims 2 - 21 , wherein the bacterial cell comprises a Type IV Secretion System (T4SS, e.g., T4ASS, (e.g., VirB/D4 system), T4BSS) or a Type III Secretion System (T3SS). 
     
     
         23 . The symbiont forming inoculum of any one of  claims 2 - 22 , wherein the bacterial cell is from a bacterial genera of  Agrobacterium  spp. (e.g.,  A. tumefaciens  (e.g., biovar 1),  A. rhizogenes  (e.g., biovar 2),  A. vitis  (e.g., biovar 3),  A. fabrum  (e.g., strain C58),  Rhizobium  spp.,  Mesorhizobium  spp.,  Sinorhizobium  spp.,  Bradyrhizobium  spp.,  Pseudomonas  spp. (e.g.,  P. savastanoi  pv.  Savastanoi ),  Phyllobacterium  spp.,  Ochrobactrum  spp.,  Azobacter  spp.,  Closterium  spp.,  Klebsiella  spp.,  Rhodospirillum  spp., or  Xanthomonas  spp. 
     
     
         24 . The symbiont forming inoculum of any one of  claims 2 - 21 , wherein the symbiont forming inoculum comprised in a cell comprises two or more cells in the form of cell culture, a plant callus, a callus culture, and/or a suspension culture. 
     
     
         25 . The symbiont forming inoculum of any one of  claims 2 - 21  or  24 , wherein the plant cell is from a macroalgae, an angiosperm, a gymnosperm or a pteridophyte. 
     
     
         26 . A symbiont comprising a plant cell comprising and expressing a polynucleotide encoding a phytohormone biosynthetic enzyme and a polynucleotide of interest, wherein the phytohormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and/or at least one auxin biosynthetic enzyme and the plant cell of the symbiont autonomously divides. 
     
     
         27 . The symbiont of  claim 26 , wherein the plant cell comprises more than one plant cell and forms an undifferentiated multi-cellular structure when transplanted onto a plant or part thereof. 
     
     
         28 . The symbiont of  claim 26  or  claim 27 , wherein the phytohormone biosynthetic enzyme is from a bacterial species and/or a plant species. 
     
     
         29 . The symbiont of any one of  claims 26 - 28 , wherein the phytohormone biosynthetic enzyme is an indole-3-acetamide hydrolase (iaaH) (E.C. Number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), a tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), an indole-3-lactate synthase (EC 1.1.1.110), a L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), a tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), a tryptophan decarboxylase 1/tryptophan decarboxylase 2 (EC4.1.1.105), an isopentenyl transferase (Ipt) and/or a Tzs (EC 2.5.1.27). 
     
     
         30 . The symbiont of any one of  claims 26 - 29 , wherein the phytohormone biosynthetic enzyme comprises indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and/or isopentenyl transferase (Ipt), optionally wherein the phytohormone biosynthetic enzyme comprises indole-3-lactate synthase. 
     
     
         31 . The symbiont of any one of  claims 26 - 30 , further comprising a polynucleotide encoding a plast polypeptide (e.g., plasticity polypeptide), optionally wherein the plast polypeptide is 6b, rolB, rolC, and/or orf13. 
     
     
         32 . The symbiont of any one of  claims 26 - 31 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest are comprised in a single nucleic acid construct or in two or more nucleic acid constructs (e.g., one or more expression cassettes). 
     
     
         33 . The symbiont of  claim 31  or  claim 32 , wherein the polynucleotide encoding a plast polypeptide is comprised in a nucleic acid construct, optionally wherein the polynucleotide encoding a plast polypeptide is in the same or a separate nucleic acid construct as the polynucleotide encoding a phytohormone biosynthetic enzyme and/or the polynucleotide of interest. 
     
     
         34 . The symbiont of any one of  claims 26 - 33 , wherein the polynucleotide of interest encodes a biomolecule, a bioactive molecule and/or a polypeptide involved in the biosynthesis of a bioactive molecule. 
     
     
         35 . The symbiont of  claim 34 , wherein expression of the polynucleotide of interest confers increased abiotic stress resistance (e.g., high salt tolerance, high heat tolerance, heavy metals tolerance, cold tolerance, drought tolerance, excessive water tolerance, tolerance to UV radiation), increased resistance or tolerance to a pathogen (e.g., viral, fungal, bacterial) or pest (e.g., insect, nematode), or increased tolerance to an herbicide. 
     
     
         36 . The symbiont of  claim 34  or  claim 35 , wherein the bioactive molecule is a pharmaceutical, a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein/peptide (e.g., a bioinsecticide, e.g., jaburetox (peptide JBTX), insecticidal peptides from spider venom (e.g., from  Hadronyche versuta ); trypsin modulating oostatic factor (TMOF),  Bacillus thuringiensis  toxins (δ endotoxins, e.g., Cry toxin, Cyt toxin), a vegetative insecticidal protein (Vip)), a nutrient (e.g., nitrogen, e.g., a leghemoglobin, a nitrogenase), a plant growth regulator (auxin, cytokinin, gibberellin, ethylene; growth inhibitor/retardant), an RNA (e.g., siRNA, dsRNA, miRNA, shRNA), a plantibody, a stylet sheath inhibitory protein (e.g., ficin, bromelain), a ribozyme, a bacteriocin, a plant lipid, a plant fatty acid, a plant oil, an antimicrobial peptide (e.g., oncocin), an aptamer, a nuclease, a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN) and/or an engineered meganuclease. 
     
     
         37 . The symbiont of any one of  claims 26 - 36 , wherein the polynucleotide of interest encodes a polypeptide operably linked to a targeting sequence, optionally wherein the targeting sequence locates the polypeptide to a membrane, a subcellular location or an extracellular location. 
     
     
         38 . The symbiont of  claim 37 , wherein the targeting sequence is a membrane targeting sequence, an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence or a plant virus movement protein. 
     
     
         39 . The symbiont of any one of  claims 26 - 38 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, a polynucleotide encoding isopentenyl transferase (Ipt), and/or a polynucleotide encoding indole-3-lactate synthase) and/or the polynucleotide encoding a plast polypeptide is/are operably linked to a nuclear targeting sequence. 
     
     
         40 . The symbiont of any one of  claims 26 - 39 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest are operably linked to a single promoter or to at least two separate promoters, in any combination. 
     
     
         41 . The symbiont of any one of  claims 26 - 40 , wherein when the polynucleotide encoding a phytohormone biosynthetic enzyme encodes iaaH, IaaM, and Ipt, the polynucleotide(s) encoding iaaH, IaaM, and Ipt are operably linked to a single promoter and the polynucleotide of interest is operably linked to the same promoter or a separate promoter. 
     
     
         42 . The symbiont of any one of  claims 26 - 41 , wherein the polynucleotide encoding a plast polypeptide is operably linked to a promoter, optionally the polynucleotide encoding a plast polypeptide is operably linked to the same promoter or a separate promoter as that which is operably linked to the polynucleotide encoding a phytohormone biosynthetic enzyme and/or the polynucleotide of interest. 
     
     
         42 . The symbiont of any one of  claims 26 - 42 , wherein the plant cell is from a macroalgae, an angiosperm, a gymnosperm or a pteridophyte. 
     
     
         43 . A host plant comprising at least one symbiont of any one of  claims 26 - 42 , wherein the at least one symbiont is located on at least one site on the host plant. 
     
     
         44 . The host plant of  claim 43 , wherein the at least one site on the host plant is on an explant, embryo, leaf, shoot, stem, branch, kernel, ear, cob, husk, stalk, epidermal tissue, apical meristem tissue, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepal, petal, receptacle, filament, style, stigma, etc), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, burl, plant food body, dormatia, extrafloral nectary, nodule, plant neoplasm or gall 
     
     
         45 . The host plant of any one of  claim 43  or  claim 44 , wherein the polynucleotide of interest is expressed in the symbiont and an expression product of the polynucleotide of interest and/or a product made using the expression product of the polynucleotide of interest is transported into the host plant. 
     
     
         46 . The host plant of any one of  claims 43 - 45 , wherein the host plant is from a wild type plant of any age or size (e.g., seedling, juvenile plant, or mature plant). 
     
     
         47 . The host plant of any one of  claims 43 - 46 , wherein the host plant is a macroalgae, an angiosperm plant, a gymnosperm plant or a pteridophyte plant. 
     
     
         48 . A method of producing a symbiont forming inoculum, the method comprising:
 introducing into a cell a polynucleotide encoding a phytohormone biosynthetic enzyme and a polynucleotide of interest or introducing a polynucleotide encoding a phytohormone biosynthetic enzyme into a transgenic cell that comprises a polynucleotide of interest, wherein the phytohormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and/or an auxin biosynthetic enzyme, thereby producing the symbiont forming inoculum, optionally, wherein the cell is a plant cell or a bacterial cell.   
     
     
         49 . The method of  claim 48 , further comprising culturing the cell to produce a population of cells comprising the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest. 
     
     
         50 . A method of producing a symbiont forming inoculum, the method comprising
 (a) (i) introducing into/onto at least one site on a plant (or a part thereof (e.g., explant)) a polynucleotide encoding a phytohormone biosynthetic enzyme and a polynucleotide sequence of interest or transplanting a plant cell comprising the same or inoculating a bacterial cell comprising the same onto at least one site on the plant (or a part thereof), or   (ii) introducing a polynucleotide encoding a phytohormone biosynthetic enzyme, or transplanting a plant cell comprising the same, or inoculating a bacterial cell comprising the same onto at least one site on the plant (or a part thereof) into/onto at least one site on a plant (or a part thereof), the plant (or a part thereof) of (ii) comprising a polynucleotide sequence of interest,   wherein the phytohormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and/or an auxin biosynthetic enzyme, thereby producing a symbiont on the plant (or part thereof) that comprises the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide sequence of interest; and   (b) selecting one or more cells from the symbiont on the plant, to provide one or more cells comprising the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide sequence of interest, thereby producing the symbiont forming inoculum.   
     
     
         51 . The method of  claim 50 , further comprising (c) culturing the one or more cells from (b) to produce a population of plant cells (e.g., a callus culture and/or a suspension culture) comprising the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide sequence of interest. 
     
     
         52 . The method of  claim 50  or  claim 51 , wherein the phytohormone biosynthetic enzyme is from a bacterial species and/or a plant species. 
     
     
         53 . The method of any one of  claims 50 - 52 , wherein the phytohormone biosynthetic enzyme is an indole-3-acetamide hydrolase (iaaH) (E.C. Number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), a tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), an indole-3-lactate synthase (EC 1.1.1.110), a L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), a tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), a tryptophan decarboxylase 1/tryptophan decarboxylase 2 (EC4.1.1.105), an isopentenyl transferase (Ipt) and/or a Tzs (EC 2.5.1.27). 
     
     
         54 . The method of any one of  claims 50 - 53 , wherein the phytohormone biosynthetic enzyme comprises indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and/or isopentenyl transferase (Ipt), and/or optionally comprises indole-3-lactate synthase. 
     
     
         55 . The method of any one of  claims 50 - 54 , wherein the at least one site on the plant is on an above ground part of the plant and/or a below ground part of the plant. 
     
     
         56 . The method of any one of  claims 50 - 55 , further comprising introducing into the cell or at least one site on a plant a polynucleotide encoding a plast polypeptide (e.g., a plasticity polypeptide), optionally wherein the plast polypeptide is 6b, rolB, rolC, and/or orf13. 
     
     
         57 . The method of any one of  claims 50 - 56 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest are introduced in a single nucleic acid construct or separately in two or more nucleic acid constructs (e.g., one or more expression cassettes). 
     
     
         58 . The method of  claim 56  or  claim 57 , wherein the polynucleotide encoding a plast polypeptide is comprised in a nucleic acid construct, optionally wherein the polynucleotide encoding the plast polypeptide is introduced in the same or a separate nucleic acid construct as the polynucleotide encoding a phytohormone biosynthetic enzyme and/or the polynucleotide of interest. 
     
     
         59 . The method of  claim 57  or  claim 58 , wherein the two or more nucleic acid constructs are comprised in one or more vectors, optionally wherein the vector is a plasmid, a T-DNA, a bacterial artificial chromosome, viral vector, or a binary-bacterial artificial chromosome. 
     
     
         60 . The method of any one of  claims 50 - 59 , wherein the polynucleotide of interest encodes a biomolecule, a bioactive molecule and/or a polypeptide involved in the biosynthesis of a bioactive molecule. 
     
     
         61 . The method of  claim 60 , wherein expression of the polynucleotide of interest confers increased abiotic stress resistance (e.g., high salt tolerance, high heat tolerance, heavy metals tolerance, cold tolerance, drought tolerance, excessive water tolerance, tolerance to UV radiation), increased resistance or tolerance to a pathogen (e.g., viral, fungal, bacterial) or pest (e.g., insect, nematode), and/or increased tolerance to an herbicide. 
     
     
         62 . The method of  claim 60  or  claim 61 , wherein the bioactive molecule is a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein/peptide (e.g., a bioinsecticide; e.g., jaburetox (peptide JBTX; a bioinsecticide derived from Jack beans ( Canavalia ensiformis  seeds)); insecticidal peptides from spider venom (e.g., from  Hadronyche versuta ); trypsin modulating oostatic factor (TMOF);  Bacillus thuringiensis  toxins (δ endotoxins, e.g., Cry toxin, Cyt toxin); a vegetative insecticidal protein (Vip)), a nutrient (e.g., nitrogen, e.g., a leghemoglobin, a nitrogenase), a plant growth regulator (auxin, cytokinin, gibberellin, ethylene; growth inhibitor/retardant), an RNA (e.g., siRNA, dsRNA, miRNA, shRNA), a plantibody, a stylet sheath inhibitory protein (e.g., ficin, bromelain), a ribozyme, a bacteriocin, an antimicrobial peptide (e.g., oncocin), a plant lipid, a plant fatty acid, a plant oil, an aptamer, a nuclease, a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN) and/or an engineered meganuclease. 
     
     
         63 . The method of any one of  claims 50 - 62 , wherein the polynucleotide of interest encodes a polypeptide operably linked to a targeting sequence, optionally wherein the targeting sequence locates the protein to a membrane, a subcellular location or an extracellular location, optionally wherein the targeting sequence is a membrane targeting sequence, an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence or a plant virus movement protein. 
     
     
         64 . The method of any one of  claims 50 - 63 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and/or the polynucleotide encoding a plast polypeptide is/are operably linked to a nuclear targeting sequence. 
     
     
         65 . The method of any one of  claims 50 - 64 , wherein the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest are each operably linked to a single promoter or to at least two separate promoters, in any combination. 
     
     
         66 . The method of any one of  claims 50 - 65 , wherein when the polynucleotide encoding a phytohormone biosynthetic enzyme encodes iaaH, IaaM, and Ipt, the polynucleotide(s) encoding iaaH, IaaM, and Ipt are operably linked to a single promoter and the polynucleotide of interest is operably linked to the single promoter or to a separate promoter. 
     
     
         67 . The method of any one of  claims 50 - 66 , wherein when the polynucleotide encoding a phytohormone biosynthetic enzyme comprises a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, and a polynucleotide encoding Ipt, the polynucleotide encoding iaaH, the polynucleotide encoding IaaM, and the polynucleotide encoding Ipt are each operably linked to at least two separate promoters and the polynucleotide of interest is operably linked to a separate promoter from the at least two separate promoters or is linked to at least one of the at least two separate promoters. 
     
     
         68 . The method of any one of  claims 56 - 67 , wherein the polynucleotide encoding a plast polypeptide is operably linked to a promoter, optionally the polynucleotide encoding a plast polypeptide is operably linked to the same promoter or a separate promoter as that which is operably linked to the polynucleotide encoding a phytohormone biosynthetic enzyme and/or the polynucleotide of interest. 
     
     
         69 . The method of any one of  claims 65 - 68 , wherein the promoter, the single promoter and/or the two or more separate promoters are a constitutive promoter or an inducible promoter. 
     
     
         70 . The method of any one of  claims 48 - 54  or  56 - 69 , wherein the bacterial cell comprises a Type IV Secretion System (T4SS, e.g., T4ASS, (e.g., VirB/D4 system), T4BSS) or a Type III Secretion System (T3SS). 
     
     
         71 . The method of any one of  claims 48 - 54  or  56 - 69 , wherein the bacterial cell is from a bacterial genera of  Agrobacterium  spp. (e.g.,  A. tumefaciens  (e.g., biovar 1),  A. rhizogenes  (e.g., biovar 2),  A. vitis  (e.g., biovar 3),  A. fabrum  (e.g., strain C58),  Rhizobium  spp.,  Mesorhizobium  spp.,  Sinorhizobium  spp.,  Bradyrhizobium  spp.,  Pseudomonas  spp. (e.g.,  P. savastanoi  pv.  Savastanoi ),  Phyllobacterium  spp.,  Ochrobactrum  spp.,  Azobacter  spp.,  Closterium  spp.,  Klebsiella  spp.,  Rhodospirillum  spp., or  Xanthomonas  spp. 
     
     
         72 . The method of any one of  claims 50 - 69 , wherein the plant, plant part or plant cell is from a wild type plant or a transgenic plant of any age or size (e.g., seedling, juvenile plant, or mature plant). 
     
     
         73 . The method of any one of  claims 50 - 69  or  72 , wherein the plant, plant part or plant cell is from a macroalgae, an angiosperm plant, a gymnosperm plant or a pteridophyte plant. 
     
     
         74 . The method of any one of  claims 50 - 69 ,  72  or  73 , wherein the plant cell is from a plant cell culture (callus culture or suspension culture), a protoplast, seedling, explant, embryo, leaf, shoot, stem, branch, kernel, ear, cob, husk, stalk, epidermal tissue, apical meristem tissue, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepal, petal, receptacle, filament, style, stigma, etc), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, burl, plant food body, dormatia, extrafloral nectary, nodule, gall or plant neoplasm. 
     
     
         75 . The method of any one of  claims 50 - 69  or  72 - 74 , wherein the at least one site on a plant is an explant, embryo, leaf, shoot, stem, branch, kernel, ear, cob, husk, stalk, epidermal tissue, apical meristem tissue, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepal, petal, receptacle, filament, style, stigma, etc), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, burl, plant food body, dormatia, extrafloral nectary, nodule, plant neoplasm or gall. 
     
     
         76 . The method of any one of  claims 50 - 75 , wherein introducing is via bacterial mediated transformation, agroinfiltration, viral-mediated transformation, particle bombardment (biolistics), electroporation, microinjection, lipofection (liposome mediated transformation), sonication, silicon fiber mediated transformation, chemically stimulated DNA uptake (e.g., polyfection; e.g., polyethylene glycol (PEG) mediated transformation), and/or laser microbeam (UV) induced transformation. 
     
     
         77 . The method of any one of  claims 50 - 69  or  72 - 76 , wherein (i) the polynucleotide encoding a phytohormone biosynthetic enzyme and a polynucleotide sequence of interest or (ii) the polynucleotide encoding a phytohormone biosynthetic enzyme is comprised in at least one plant cell, and the at least one plant cell is transplanted onto the at least one site on the plant. 
     
     
         78 . The method of  claim 77  wherein the plant is wounded at the at least one site prior to or during transplanting of the at least one plant cell to the at least one site on the plant. 
     
     
         79 . The method of  claim 76 , wherein the introducing is via bacterial mediated transformation and comprises co-cultivating the plant cell or plant (or a part thereof, e.g., explant) with the cells of at least one bacterial species or strain, the bacterial cells comprising one or more of: the polynucleotide encoding a phytohormone biosynthetic enzyme, the polynucleotide of interest, and/or at least one polynucleotide encoding at least one plast polypeptide. 
     
     
         80 . The method of  claim 79 , wherein the plant (or part thereof; e.g., explant) is wounded at the at least one site prior to or during co-cultivation with the cells of the at least one bacterial strain. 
     
     
         81 . The method of  claim 79  or  claim 80 , wherein the cells of the at least one bacterial species or strain comprise cells of at least two bacterial species or strains and the polynucleotide encoding the at least one phytohormone enzyme is comprised in a separate bacterial strain from the bacterial strain comprising the at least one polynucleotide of interest (e.g., dual bacterial transformation). 
     
     
         82 . The method of any one of  claims 79 - 81 , wherein the at least one bacterial strain or species and/or the at least two bacterial strains or species is/are a bacterial cell comprising a Type IV Secretion System (T4SS, e.g., T4ASS, (e.g., VirB/D4 system), T4BSS) or a Type III Secretion System (T3SS). 
     
     
         83 . The method of  claim 82 , wherein the bacterial cell is an  Agrobacterium  spp. cell (e.g.,  A. tumefaciens  (e.g., biovar 1),  A. rhizogenes  (e.g., biovar 2),  A. vitis  (e.g., biovar 3),  A. fabrum  (e.g., strain C58),  Rhizobium  spp. cell,  Mesorhizobium  spp. cell,  Sinorhizobium  spp. cell,  Bradyrhizobium  spp. cell,  Pseudomonas  spp. cell (e.g.,  P. savastanoi  pv.  Savastanoi ),  Phyllobacterium  spp. cell,  Ochrobactrum  spp. cell,  Azobacter  spp. cell,  Closterium  spp. cell,  Kiebsiella  spp. cell,  Rhodospirillum  spp. cell, or  Xanthomonas  spp. cell. 
     
     
         84 . The method of any one of  claims 48 - 83 , the method further comprising editing of at least one nucleic acid in at least one cell of the symbiont forming inoculum to produce at least one edited nucleic acid, optionally wherein the editing is carried out with a gene editing nuclease. 
     
     
         85 . The method of  claim 84 , wherein the at least one edited nucleic acid has modified expression (e.g., increased or decreased expression as compared to the same nucleic acid not so modified). 
     
     
         86 . A symbiont forming inoculum produced by the method of any one of  claims 48 - 85 . 
     
     
         87 . The symbiont forming inoculum of  claim 86 , wherein the inoculum comprises a bacterial cell, a bacterial culture, a plant cell, or a plant cell culture (e.g., a callus or a cell suspension). 
     
     
         88 . A cell or protoplast from the symbiont forming inoculum of  claim 86  or  claim 87 , wherein the cell or protoplast comprises the polynucleotide encoding a phytohormone biosynthetic enzyme and the polynucleotide of interest. 
     
     
         89 . A method of modifying a host plant characteristic without modifying the plant genome, the method comprising
 transplanting the symbiont forming inoculum of  claim 86  or  claim 87  or cell of  claim 88 , or the symbiont of any one of  claims 26 - 42  to at least one site on a host plant; and   culturing the symbiont forming inoculum or symbiont at the at least one site on the host plant to form a symbiont on the host plant at the at least one site, wherein the polynucleotide of interest is expressed in the symbiont on the host plant and an expression product of the polynucleotide of interest and/or a product made using the expression product of the polynucleotide of interest is transported into the host plant, thereby producing a host plant having a modified characteristic.   
     
     
         90 . The method of  claim 89 , wherein the polynucleotide of interest encodes a biomolecule, a bioactive molecule and/or a polypeptide involved in the biosynthesis of a bioactive molecule. 
     
     
         91 . The method of  claim 90 , wherein expression of the polynucleotide of interest confers increased abiotic stress resistance (e.g., high salt tolerance, high heat tolerance, heavy metals tolerance, cold tolerance, drought tolerance, excessive water tolerance, tolerance to UV radiation), increased resistance or tolerance to a pathogen (e.g., viral, fungal, bacterial) or pest (e.g., insect, nematode), or increased tolerance to an herbicide 
     
     
         92 . The method of  claim 90  or  claim 91 , wherein the bioactive molecule is a pharmaceutical (e.g., a therapeutic protein, a therapeutic polynucleotide, a therapeutic chemical; e.g., a vaccine, an antibody, a recombinant antibody, an antibody fragment, a fusion protein, an antibody fusion protein, human serum albumin, gastric lipase, insulin, glucocerebrosidase, growth factor, a cytokine, hepatitis B surface antigen (HBsAg)), Apo-A1, alpha-galactosidase (PRX-102), alpha-galactosidase (PRX-102), acetylcholineesterase (PRX-105), antitumor necrosis factor (Pr-anti-TNF), IgG, interferon-alpha, plasmin, lactoferrin, lysozyme, and collagen), a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein/peptide (e.g., a bioinsecticide; e.g., jaburetox (peptide JBTX; a bioinsecticide derived from Jack beans ( Canavalia ensiformis  seeds), insecticidal peptides from spider venom (e.g., from  Hadronyche versuta ); trypsin modulating oostatic factor (TMOF),  Bacillus thuringiensis  toxins (δ endotoxins, e.g., Cry toxin, Cyt toxin); a vegetative insecticidal protein (Vip), a nutrient (e.g., nitrogen, e.g., a leghemoglobin, a nitrogenase), a plant growth regulator (auxin, cytokinin, gibberellin, ethylene; growth inhibitor/retardant), an RNA (e.g., siRNA, dsRNA, miRNA, shRNA), a plantibody, a plant lipid, a plant fatty acid, a plant oil, a stylet sheath inhibitory protein (e.g., ficin, bromelain), a ribozyme, a bacteriocin, an antimicrobial peptide (e.g., oncocin), an aptamer, a nuclease, a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN) and/or an engineered meganuclease. 
     
     
         93 . The method of  claim 89 - 92 , wherein the at least one site on a host plant is on an above ground part of the host plant and/or on a below ground part of the host plant. 
     
     
         94 . The method of any one of  claims 89 - 91 , wherein the symbiont or the symbiont forming inoculum is transplanted onto the host plant at least two times and/or onto at least two sites on the host plant. 
     
     
         95 . The method of any one of  claims 89 - 94 , wherein the expression product is a transcription product or a translation product, or a modification thereof. 
     
     
         96 . The method of any one of  claims 89 - 95 , wherein the product made using the expression product of the polynucleotide is a chemical, a protein (polypeptide/peptide) or a polynucleotide. 
     
     
         97 . The method of any one of  claims 89 - 96 , wherein the modified host plant characteristic is increased tolerance/resistance to a disease causing organism (e.g., a fungus, a bacteria, a virus). 
     
     
         98 . The method of any one of  claims 89 - 97 , wherein the modified host plant characteristic is increased (induced) expression of plant defense genes. 
     
     
         99 . The method of any one of  claims 89 - 98 , wherein the modified host plant characteristic is increased insect tolerance/resistance. 
     
     
         100 . The method of any one of  claims 89 - 99 , wherein the modified host plant characteristic is increased nematode tolerance/resistance. 
     
     
         101 . The method of any one of  claims 89 - 100  wherein the modified host plant characteristic is a modified morphology. 
     
     
         102 . The method of  claim 101 , wherein the modified morphology comprises shortened internodes, increased lateral branching and/or increased flowering. 
     
     
         103 . The method of any one of  claims 89 - 102 , wherein the modified host plant characteristic is the presence of a biomolecule, a bioactive molecule and/or a polypeptide involved in the biosynthesis of a bioactive molecule. 
     
     
         104 . The method of  claim 103 , wherein the bioactive molecule is a pharmaceutical (e.g., a therapeutic protein, a therapeutic polynucleotide, a therapeutic chemical), a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein/peptide, a trypsin modulating oostatic factor (TMOF); a  Bacillus thuringiensis  toxin, a vegetative insecticidal protein (Vip), a nutrient, a plant growth regulator, an RNA, a plantibody, a stylet sheath inhibitory protein, a ribozyme, a bacteriocin, an antimicrobial peptide, a plant lipid, a plant fatty acid, a plant oil, an aptamer, a nuclease, a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN) and/or an engineered meganuclease. 
     
     
         105 . The method of  claim 103  or  104 , wherein the bioactive molecule is jaburetox (peptide JBTX) an 5 endotoxin, a Cry toxin, a Cyt toxin, a leghemoglobin, a nitrogenase, ficin, bromelain a bacteriocin, nisin and/or oncocin). 
     
     
         106 . The method of any one of  claims 89 - 105 , wherein the modified host plant characteristic is the presence of a bioactive molecule (e.g., biocidal molecule) in the host plant and increased resistance/tolerance to a plant pathogen. 
     
     
         107 . The method of 106, wherein the bioactive molecule is a bacteriocin or an antimicrobial peptide and the plant pathogen is a bacterium. 
     
     
         108 . The method of  claim 107 , wherein the bacteriocin or antimicrobial peptide is oncocin and/or nisin. 
     
     
         109 . The method of any one of  claims 89 - 105 , wherein the modified host plant characteristic is the presence of an insecticidal protein (e.g., a bioinsecticide) and increased insect tolerance or resistance. 
     
     
         110 . The method of  claim 109 , wherein the insecticidal protein is jaburetox, trypsin modulating oostatic factor (TMOF), a  Bacillus thuringiensis  toxin (e.g., δ endotoxins, e.g., Cry toxin, Cyt toxin); a stylet sheath inhibitory protein (e.g., ficin, bromelain), and/or a vegetative insecticidal protein (Vip). 
     
     
         111 . The method of any one of  claims 89 - 104 , wherein the modified host plant characteristic is the presence of or increased or decreased production of a plant growth regulator (e.g., auxin, cytokinin, gibberellin, ethylene; a growth inhibitor/retardant) and modified growth. 
     
     
         112 . The method of any one of  claims 89 - 104 , wherein the modified host plant characteristic is the presence of or increased production of an RNA and increased/decreased production of a polynucleotide, a peptide or a polypeptide. 
     
     
         113 . The method of  claim 112 , wherein the RNA is a siRNA, a dsRNA, a miRNA, or a shRNA, optionally dvsnf7, ccomt, dCS, asn1, phL, RI, PGAS, and/or ppo5. 
     
     
         114 . A host plant having a modified characteristic produced by the method of any one of  claims 89  to  113 . 
     
     
         115 . A method of producing a biomolecule and/or bioactive molecule, comprising
 providing the symbiont of any one of  claims 26 - 42 , wherein the polynucleotide of interest encodes a biomolecule and/or bioactive molecule and collecting the biomolecule and/or bioactive molecule produced in the symbiont; and/or   providing the host plant of any one of  claims 43 - 47 , wherein the polynucleotide of interest encodes a biomolecule and/or bioactive molecule and collecting the biomolecule and/or bioactive molecule produced in the symbiont and host plant.   
     
     
         116 . A method of delivering a compound of interest to a host plant, comprising
 transplanting the symbiont forming inoculum of any one of  claims 1 - 25 ,  86 , or  87 , or the cell of  claim 88  or the symbiont of any one of  claims 26 - 42  onto at least one site on a host plant; and   culturing the symbiont forming inoculum or symbiont at the at least one site on the host plant to form a symbiont on the host plant at the at least one site, wherein the polynucleotide of interest is expressed in the symbiont and an expression product of the polynucleotide of interest and/or a product made using the expression product of the polynucleotide of interest is transported into the host plant, thereby delivering the compound of interest to a plant.   
     
     
         117 . A method of producing a plant comprising a modified characteristic without modifying the plant's genotype, comprising:
 transplanting the symbiont forming inoculum of any one of  claims 1 - 25 ,  86 , or  87 , or the cell of  claim 88 , or the symbiont of any one of  claims 26 - 42  onto at least one site on a host plant; and   culturing the symbiont forming inoculum or symbiont at the at least one site on the host plant to form a symbiont on the host plant at the at least one site, wherein the polynucleotide of interest is expressed in the symbiont and an expression product of the polynucleotide of interest and/or a product made using the expression product of the polynucleotide of interest is transported into the host plant, thereby producing the plant comprising a modified characteristic without a modified genotype.   
     
     
         118 . A plant produced by the method of  claim 117 .